We propose to investigate the mechanisms of the anti-viral innate immune response using single molecule fluorescence techniques in vitro and super-resolution imaging of cells. RIG-I (retinoic acid inducible gene-l) and related receptors were recently identified as the initial sensors for viral RNA that signal downstream molecules. RIG-I has a central DExD/H RNA helicase domain. The N-terminus possesses tandem CARDs (caspase activation and recruitment domains) that interact with a mitochondrial antiviral signaling protein (MAVS) and are ubiquitinated by TRIM25 E3 ligase. It also has a C-terminal regulatory domain (RD) that senses 5' triphosphate which is the primary signature of viral RNA. RIG-I also recognizes double stranded (ds) RNA as a viral signature. RIG-I is an RNA-dependent ATPase and its ATPase activity closely correlate with its signaling function. However, the role of its ATPase activity has remained a mystery. Using proteininduced fluorescence enhancement (PIPE) at the single molecule level in collaboration with Project 2. we found that RIG-I translocates rapidly and repeatedly on short dsRNA (20-50 bp). We also showed that its movement is slowed down by CARDs and accelerated by the presence of 5' triphosphate. Combined with previous studies that show a strong correlation between ATPase activity and RIG-I signaling, this data indicates that ATP-powered RNA translocation is essential for RIG-I signaling. In Project 3, we will address many of the outstanding questions such as how RIG-I discriminates between viral RNA and similar-looking host RNA molecules, what the role of RIG-l's ATPase activity is, how RIG-l's function is regulated by nucleic acid composition, what its interactions with other proteins are and how RIG-l's post-transcriptional and posttranslational modifications affect its function. There are three specific aims: In Aim 1, we will investigate the RNA translocation activities of RIG-I like receptors. In Aim 2, we will investigate RIG-I loading and oligomerization, and its conformational changes upon viral RNA recognition. In Aim 3, we will investigate cellular location of RIG-I and its partners such as MAVS and viral RNA using live cell imaging and super-resolution imaging. In addition, RIG-I interaction with MAVS will be studied at the single molecule level in a reconstituted system.